The gene old of bacteriophage P2 is known to (i) cause interference with phage A growth; (ii) kill ...... Calendar, R., B. Lindqvist, G. Sironi, and A. J. Clark. 1970.
Vol. 48, No. 3
JOURNAL OF VIROLOGY, Dec. 1983, p. 616-626 0022-538X/83/120616-11$02.00/0 Copyright ©D 1983, American Society for Microbiology
An Escherichia coli Gene Required for Bacteriophage P2-K Interference DANIELA GHISOTTI, SANDRO ZANGROSSI, AND GIANPIERO SIRONI* Dipartimento di Biologia, Sezione di Genetica e Microbiologia, Universitd di Milano, 20133 Milan, Italy
Received 24 March 1983/Accepted 17 August 1983
The gene old of bacteriophage P2 is known to (i) cause interference with phage A growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the abovementioned phenomena caused by the old' gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated. The gene old of bacteriophage P2 has the following properties: (i) it interferes with A growth after A infection of P2 lysogens or A induction of X,P2 double lysogens (27); (ii) it kills recB- mutants of Escherichia coli after P2 infection (27, 37); (iii) it causes increased sensitivity of P2 lysogens to X-ray irradiation (19). The P2-K interference phenomenon has been extensively investigated. In the presence of P2 in the host cell, K DNA is poorly replicated, and only K early functions are expressed (27, 38). At the same time, severe inhibition of host protein and RNA syntheses and cell killing occur (9, 10, 38). The shutoff of protein synthesis has been found to be related to the inactivation of several transfer RNA species (9). The inhibition of protein synthesis and tRNA inactivation depend on several elements required for K DNA replication: the K genes 0 and P, the K replication origin, and the host dnaB product (10, 11). K mutants able to escape from P2 interference are called Spi- and carry a Chi recombinational hot spot and two mutations, one in the Red function, which promotes general recombination, and the other in gene gam, which codes for an inhibitor of bacterial exonuclease V (25, 27, 30, 38, 49). A striking similarity exists between P24 interference and the other phenomena caused by P2 old': infection with P2 of recB- mutants or Xray irradiation of P2 lysogenic cells lead to inhibition of host macromolecular syntheses, above all host protein synthesis (19, 38). The mechanisms leading to cell killing and to inhibition of protein synthesis, both in P2-X interference and after P2 infection of a recB- host, have at least one step in common, since the product of 616
the K gene gam inhibits DNA exonuclease V, the product of recB and recC genes (33, 38, 46). A further suggestion for a common mechanism is given by the effect of a mutation in the host gene sbcA, which derepresses the synthesis of exonuclease VIII (2, 24). Such a mutation suppresses the incompatibility between a recBmutation and the presence in the cell of P2 old' (11, and our unpublished data) and suppresses P24 interference (11). However, the high X-ray sensitivity of P2 lysogens is not reduced by the sbcA mutation (unpublished data). Bacterial mutants able to suppress the effects of the gene old have been isolated. These mutants identify at least one new gene. MATERIALS AND METHODS Bacterial strains. The bacterial strains used are listed in Table 1. Derivatives of E. coli C (47) and E. coli K12 (1) were used. Hybrid strains between E. coli strains C and K-12 were constructed. Thy- mutants were selected with trimethoprim (32). Strains carrying F+ or F' were screened by plating the male-specific phage p.2. The presence of a recB- mutation was tested by the UV sensitivity of the strain. In crosses or transduction experiments, pin was moved as unselected marker and screened by plating X in the P2 lysogenic derivative. Phage strains. The X mutants bioll (31), bio69 (31), bio72 (44), Spil4 (19) and c1857 (43); the P2 mutants virl (8), vir3 (45), oldl (37), Aaml29 (26), and cai (7); and the P4 mutants virl (28) and oaml (20) were used. P1 k c cc (12) was used for transduction experiments. Phage PL2 (16) was used for screening of F+ or F' strains. P2 Hyl dis (large plaques) (14) was used for curing of P2 prophage. Media and chemicals. LC broth (15), EM9, and EDavis minimal media (19) were used. The media were
VOL. 48, 1983
E. COLI MUTANTS AFFECTED IN P2-X INTERFERENCE
supplemented with 10 mM MgSO4 for X adsorption and with 5 mM CaCl2 for P2 or P4 adsorption. [3H]phenylalanine was purchased from The Radiochemical Centre, Amersham, England; mitomycin C was from Sigma Chemical Co., St. Louis, Mo. Isolation ofpin- mutants. Bacterial mutants lysogenic for P2 old' and supporting growth of X Spi+ were searched. Strain C-2529, carrying two P2 old' prophages, was used. Under these conditions, the chance that mutations in each of the prophage old genes would allow growth of X Spi+ is very low. Cells of strain C-2529 were treated with nitrosoguanidine (250 ,ug/ml) for 20 min, washed, and incubated overnight. Samples were plated, and colonies were spot tested for their ability to support growth of X Spi+. Four colonies of 8,000 tested in three mutagenic treatments were found to plate X Spi+ and still to carry at least one P2 old' prophage, as shown by its ability to interfere with A growth when transferred to a wild-type strain. Three of these clones were cured of prophage by infection with P2 Hyl dis (39) and lysogenized again with P2 old'; they were still found to plate A Spi+. On this basis, they were assumed to carry a mutation in the bacterial chromosome, named pin (P2 interference). Two of these mutants were obtained in the same mutagenic treatment and yet were assumed to be of independent origin since each carried an additional and different growth requirement (for cysteine and for serine associated with pin-I and pin-3, respectively). Further characterization of the pin- mutations showed that pin-i and pin-3 differed in phenotype (see below). Construction of pin- recB- strains. Thy- derivatives of CK-2718 (pin-i), CK-2722 (pin-3), and C-la (pin') were obtained by selection with trimethoprim (32). Alternatively, the pin-i or the pin-3 mutations were introduced into strain CK-2792 (purE, thy) by transduction, selecting for the Pur+ phenotype. P2 lysogenic derivatives of the selected transductants were screened for the unselected marker pin. The recB21 mutation of strain JC4695 was introduced into each of these strains by Pl transduction, selecting for Thy' recombinants, and the recB- transductants were identified by their increased UV sensitivity (17). Dominance test. Strain W3747 containing F'13 (F' lac+lmet-) and ORF4/KL251 containing F'254 (F' lac+lleu- met- trp-) were mated with strain CK-2728 (lac- pin-i) and CK-2729 (lac- pin-3), and lac+ met' and lac+ leu+ met' trp+ recombinants were selected, respectively. Ten purified recombinants from each conjugation were analyzed for the presence of the F' by ascertaining their sensitivity to the male-specific phage >2 and for the Pin' phenotype by ascertaining their inability to plate A+ when lysogenic for P2. X-ray irradiation of bacteria. Bacterial suspensions (2 x 107 cells per ml) were irradiated with a Machlett OEG-60 X-ray tube, with 40 kilorads per min at 50 kV and 30 mA. Protein synthesis. Cells were grown with aeration in EM9 medium to 2 x 108 cells per ml. In the different experiments, samples were either shifted to 42°C to induce XcI857, irradiated with different X-ray doses, or infected with P2 or P4 mutants at a multiplicity of infection of 10. The samples were incubated with the addition of 5 ,uCi of L-[3H]phenylalanine (specific activity, 1 Ci/mmol). At intervals, incorporation of [3H]phenylalanine into acid-insoluble material was measured.
617
RESULTS The interference between P2 and A requires functions coded by each of these phages (10, 27, 49). We have looked for bacterial genes necessary for P2-X interference and unessential for bacterial growth. Bacterial mutants lysogenic for P2 old' in which A Spi+ could grow have been selected as described above. Four such mutants were isolated from strain C-2529 and the mutations named pin (P2 interference). Mapping ofpin mutations. Preliminary crosses involving E. coli C strains indicated that two of the pin mutations (pin-i and pin-3) were linked to the lac-gal region. The other two (pin-2 and pin4) do not map in the same region and were not investigated further. To proceed to a more detailed mapping of pin-i and pin-3, since in the lac-gal region of E. coli C few markers are available, E. coli K-12 markers were transferred into E. coli C by conjugation. The hybrid strains thus obtained were used as recipients in Pl transduction experiments, and the cotransduction of pin- mutations with the markers listed in Fig. 1 was studied. The pin mutations are cotransducible with high frequency with purE and with a frequency of 1% or less with dnaZ (Table 2). No cotransduction of pin was found with lac, proC, or tsx (data not shown). The cotransduction frequency of dnaZ and purE was much higher than the cotransduction of dnaZ and pin (Table 2). Furthermore, the frequency of cotransduction of pin- with the two markers dnaZ+ purE+ in double selection experiments was lower than the frequency obtained by selecting for purE+ alone. Thus, pin-i and pin-3 are likely to map distal to purE, relative to dnaZ, suggesting the order dnaZ, purE, pin. Since the markers used for the mapping of these pin mutations belong to a chromosomal region derived from E. coli K-12, we conclude that these pin mutations map at 12 min on the current E. coli K-12 map (1). However, unseen complications due to the use of hybrid strains cannot be excluded. The pin-i or the pin-3 mutations or their wild-type allele were then transferred by transduction as unselected markers into the same strain, and the strains obtained (CK-2718, CK-2722, and CK-2724, respectively) were used for further characterization. Pin- mutations are recessive. Plasmids F'13 and F'254 contain the region of the chromosome in which pin-i and pin-3 are located (18). These plasmids were transferred into strains carrying the pin-i or the pin-3 mutation (see above). In all of the clones tested, the introduction of the F' factors restored the Pin+ phenotype. Lacsegregants that had lost the F' factor were Pin-. Thus, pin-i and pin-3 are recessive. P2-X interference does not occur in pin- mutants. X+ plates efficiently on pin-l(P2) or pin-3-
618
GHISOTTI, ZANGROSSI, AND SIRONI
J. VIROL.
TABLE 1. Bacterial strains Bacterial no.
Relevant genotypea
Source or reference
E. coli C derivatives
C-la C-85 C-520 C-1058 C-1100 C-1675 C-2529 C-2531
Prototroph F+ str-2 F+ supD Prototroph (P2) (P2 cai) his-i leu-6 lyd-i str-i thr-S xan-J thy-37 thy-30 (P2) (P2 cai) cys-10 pin-i thy-30 (P2)
C-2533
pin-3 ser-10 thy-30 (P2)
C-2539
cys-10 pin-i thy-30
C-2541
pin-3 ser-10 thy-30
C-2542 C-2543 C-2565
cys-10 pin-i thy-30 (P2) pin-3 ser-10 thy-30 (P2) pin-3
C-2566 C-2617 C-2686 C-2698 C-2771
pin-3 (P2) F+ cys-10 pin-i thy-30 (P2) F+ pin-3 ser-10 thy-30 (P2) gal-30 recB21
E. coli K-12 derivatives AX727
JC4695 KG-6 KG-12 KG-14
dnaZ gal lac str thi recB21 ara lac leu proC purE str supE42 thi trp tsx xyl (P2) F+ ara lac leu proC purE str supE42 thi trp tsx xyl (P2) lac leu pin-i proC supE42 trp tsx str (P2)
KG-18 KG-21
dnaZ gal lac str thi (P2) dnaZ lac purE str thi trp tsx (P2)
ORF4/KL251
F'254 lac+--lip+/multiply marked strain carrying leu met trp F'13 dnaH+ lac+ leu+ purE+ tsx+lmet thi str ara lac leu proC purE str supE42 thi trp tsx xyl
W3747
X478G3
34 23 42 27 37 From C-la, selection with trimethoprim From C-1058, selection with trimethoprim From C-2529 by NTG treatment; not tested for the presence of a second P2 prophage From C-2529 by NTG treatment; not tested for the presence of a second P2 prophage From C-2531, cured of P2 prophage by infection with P2 Hyl dis From C-2533, cured of P2 prophage by infection with P2 Hyl dis From C-2539 by lysogenization From C-2541 by lysogenization By P1 transduction: donor C-1100; recipient C-2541; selection Thy' From C-2565 by lysogenization From a cross C-85 x C-2542 From a cross C-520 x C-2543 From C-la by UV treatment By P1 transduction: donor JC4695; recipient C-1675; selection Thy'
18 27 From X478G3 by lysogenization From a cross C-520 x KG-6
By P1 transduction: donor C-2617; recipient KG-6; selection Pur+; not tested for ara, thi, xyl From AX727 by lysogenization From a cross KG-12 x KG-18; not tested for ara, sup, xyl 6 18 21
E. coli C x K-12
hybrids CK-2710
purE str supE42 tsx
CK-2718
pin-lb supE42
CK-2722
pin-3b supE42
CK-2723c CK-2724
pin-3 supE42 supE42
CK-2726 CK-2727 CK-2728 CK-2729
lac-31 pin-i supE42 lac-32 pin-3 supE42 lac-31 pin-i supE42 (P2) lac-32 pin-3 supE42 (P2)
From a cross KG-12 x C-2698; not tested for ara, thi, xyl By Pl transduction: donor C-2617; recipient CK-2710; selection Pur+ By P1 transduction: donor C-2566; recipient CK-2710; selection Pur+ From CK-2722 by lysogenization By Pl transduction: donor C-2566; recipient CK-2710; selection for Pur+ From CK-2718, spontaneous mutant From CK-2722, spontaneous mutant From CK-2726 by lysogenization From CK-2727 by lysogenization
E. COLI MUTANTS AFFECTED IN P2-X INTERFERENCE
VOL. 48, 1983
619
TABLE 1-Continued Relevant genotype"
Bacterial no.
CK-2730 CK-2731 CK-2735 CK-2736 CK-2737 CK-2752 CK-2753 CK-2759
pin-i supE42 (P2) pin-i supE42 (P2 oldl) pin-3 supE42 (P2 oldl) supE42 (P2) supE42 (P2 oldl) pin-i supE42 thy-31 pin-3 supE42 thy-32 pin-I recB21 supE42
CK-2765
pin-3 recB21 supE42
CK-2786
purE
CK-2792 CK-2795
pin-i' thy-33
CK-2797
pin-3b thy-33
CK-2798
thy-33
CK-5004
pin-i recB21
CK-5008
pin-i recB21
CK-5009
pin-3
CK-5011
pin'
KG-9
pin-3 str trp tsx (P2)
Source or reference
From CK-2718 by lysogenization From CK-2718 by lysogenization From CK-2722 by lysogenization From CK-2724 by lysogenization From CK-2724 by lysogenization From CK-2718; selection with trimethoprim From CK-2722; selection with trimethoprim By Pl transduction: donor JC4695; recipient CK-2752; selection Thy' By P1 transduction: donor JC4695; recipient CK-2753; selection Thy' From a cross KG-12 x C-2698. Not tested for ara, thi, xyl From CK-2786; selection with trimethoprim By P1 transduction: donor C-2617; recipient CK-2792; selection Pur+ By Pl transduction: donor C-2617; recipient CK-2792; selection Pur+ By P1 transduction: donor C-2617; recipient CK-2792; selection Pur+ By P1 transduction: donor JC4695; recipient CK-2795; selection Thy' By P1 transduction: donor JC4695; recipient CK-2797; selection Thy' By P1 transduction: donor JC4695; recipient CK-2797; selection Thy' By P1 transduction: donor JC4695; recipient CK-2798; selection Thy' From a cross C-2686 x KG-6; not tested for ara, thi, sup, xyl
purE thy-33
a The markers are listed in alphabetical order. bThe presence of the pin- mutation was tested by plating A on the P2 c Strain CK-2723 is the same as C-2723 in reference 15.
(P2) (Table 3), showing that P2-A interference does not occur in pin- mutants. As expected, Xbioll, which has a Spi- phenotype (38), plates well on pin' or pin- strains lysogenic for P2; Abio72, instead, which is red- gam+ and does not plate on pin+(P2), plates well on pin-3(P2), but with reduced efficiency on pin-i(P2). The pin-i mutation is not directly responsible for the lower efficiency of plating, since Xbio72 plates well on the pin-i strain nonlysogenic for P2 or lysogenic for P2 old-. Similar results were obtained with Xbio69 or with the point mutant red3 (data not shown). Thus, the pin-i mutation does not completely restore the ability to plate in the 4+ -+ rC_ _- _ rL ola gami. ior A rea presence otrC 'not X growth was also measured after heat inducttion of X in pin-(XcI857)(P2) and after infection with X of pin-(P2) (data not shown). The frac1
purE pin . , . II,l 11 13 12 10 FIG. 1. Position of pin on the genetic map of E. coli. The positions of genes other than pin are from Ireference 1. lac proC tsx . I. I 1, I 1 3 9
dnaZ
lysogenic derivative.
tion of infected cells yielding phage (yielder frequency) and phage production are similar in the pin mutants, whether the strain is lysogenic TABLE 2. Transductional mapping of pin-i and pin-3a Cotransduction frequency with the
dnaZ+
selected marker of the following unselected marker: pin-3 pin-I purE+ dnaZ+ 0.01
